Adenosine triphosphate synthase (F1F0 ATP synthase) is the central enzyme for energy transduction in virtually all organisms. Deficiencies in energy transduction cause a group of tragic inheritable human disorders known collectively as mitochondrial myopathies. It is likely that less deleterious problems in energy metabolism will remain undetected in the clinical laboratory problems in energy metabolism will remain undetected in the clinical laboratory while acting as a contributing factor in many other health difficulties such as heart disease. Altered energy metabolism is also found in most rapidly growing tumors. F1F0 ATP synthases function by utilizing the energy of proton translocation across biological membrane to drive the phosphorylation of ADP making ATP. The long-term objective of the research is to obtain an understanding of proton movement through the enzyme at the molecular level. Molecular genetics and biochemistry are used as a unified approach. Mutations in the Escherichia coli F1F0 ATP synthase are generated by site-directed mutagenesis and the effects of the mutations investigated by a battery of biochemical analyses. In this way, the functional roles of specific amino acids within the enzyme in proton conductivity can be assessed. Difficulties associated with studying hydrophobic proteins via traditional biochemical approaches has frustrated attempts to gain a structural model of the proton translocation domain in F1F0 ATP synthase. An alternate approach using molecular biology to insert chemically reactive target amino acids into the membrane-bound portion of the enzyme is employed. The environment of the target amino acid can then be probed by established methods which could not be used previously. Finally, methods for testing hypotheses generated by experimentation with the bacterial F1F0 ATP synthase in the enzymes of higher organisms are explored. Specific approaches include the construction of a hybrid bacterial enzyme containing the human proton translocation system and the investigation of the proton translocation system of the nearest bacterial relative of the mammalian mitochondrial enzyme, Rhodobacter sphaeroides.